Tumor Necrosis Factor (TNF-alpha) and Lymphotoxin (TNF-beta) (hereinafter, TNF, refers to both TNF-alpha and TNF-beta) are multifunctional pro-inflammatory cytokines formed mainly by mononuclear phagocytes, which have many effects on cells (Wallach, D. (1986) In: Interferon 7 (Ion Gresser, ed.), pp. 83-122, Academic Press, London; and Beutler and Cerami (1987)). Both TNF-alpha and TNF-beta initiate their effects by binding to specific cell surface receptors. Some of the effects are likely to be beneficial to the organism: they may destroy, for example, tumor cells or virus infected cells and augment antibacterial activities of granulocytes. In this way, TNF contributes to the defense of the organism against tumors and infectious agents and contributes to the recovery from injury. Thus, TNF can be used as an anti-tumor agent in which application it binds to its receptors on the surface of tumor cells and thereby initiates the events leading to the death of the tumor cells. TNF can also be used as an anti-infectious agent.
However, both TNF-alpha and TNF-beta also have deleterious effects. There is evidence that overproduction of TNF-alpha may play a major pathogenic role in several diseases. For example., effects of TNF-alpha, primarily on the vasculature, are known to be a major cause for symptoms of septic shock (Tracey et al, 1986). In some diseases, TNF may cause excessive loss of weight (cachexia) by suppressing activities of adipocytes and by causing anorexia, and TNF-alpha was thus called cachectin. It was also described as a mediator of the damage to tissues in rheumatic diseases (Beutler and Cerami, 1987) and as a major mediator of the damage observed in graft-versus-host reactions (Piquet et al, 1987). In addition, TNF is known to be involved in the process of inflammation and in many other diseases.
Two distinct, independently expressed, receptors, the p55 (CD120a) and the p75 (CD120b) TNF-Rs, which bind both TNF-alpha and TNF-beta specifically, initiate and/or mediate the above noted biological effects of TNF. These two receptors have structurally dissimilar intracellular domains suggesting that they signal differently (See Hohmann et al, 1989; Engelmann et al, 1990; Brockhaus et al, 1990; Loetscher et al, 1990; Schall et al, 1990; Nophar et al, 1990; Smith et al, 1990; and Holler et al, 1990). However, the cellular mechanisms, for example, the various proteins and possibly other factors, which are involved in the intracellular signaling of the CD120a and CD120b have yet to be elucidated. It is intracellular signaling, which occurs usually after the binding of the ligand, i.e., TNF (alpha or beta), to the receptor, that is responsible for the commencement of the cascade of reactions that ultimately result in the observed response of the cell to TNF.
As regards the above-mentioned cytocidal effect of TNF, in most cells studied so far, this effect is triggered mainly by CD120a. Antibodies against the extracellular domain (ligand binding domain) of CD120a can themselves trigger the cytocidal effect (see EP 412486) which correlates with the effectiveness of receptor cross-linking by the antibodies, believed to be the first step in the generation of the intracellular signaling process. Further, mutational studies (Brakebusch et al, 1992; Tartaglia et al, 1993) have shown that the biological function of CD120a depends on the integrity of its intracellular domain, and accordingly it has been suggested that the initiation of intracellular signaling leading to the cytocidal effect of TNF occurs as a consequence of the association of two or more intracellular domains of CD120a. Moreover, TNF (alpha and beta) occurs as a homotrimer, and as such, has been suggested to induce intracellular signaling via CD120a by way of its ability to bind to and to cross-link the receptor molecules, i.e., cause receptor aggregation.
Another member of the TNF/NGF superfamily of receptors is the FAS/APO1 receptor (CD95), which has also been called the FAS antigen, a cell-surface protein expressed in various tissues and sharing homology with a number of cell-surface receptors including TNF-R and NGF-R. CD95 mediates cell death in the form of apoptosis (Itoh et al, 1991), and appears to serve as a negative selector of autoreactive T cells, i.e., during maturation of T cells, CD95 mediates the apoptotic death of T cells recognizing self-antigens. It has also been found that mutations in the CD95 gene (lpr) cause a lymphoproliferation disorder in mice that resembles the human autoimmune disease systemic lupus erythematosus (SLE) (Watanabe-Fukunaga et al, 1992). The ligand for CD95 appears to be a cell-surface associated molecule carried by, amongst others, killer T cells (or cytotoxic T lymphocytes—CTLs), and hence when such CTLs contact cells carrying CD95, they are capable of inducing apoptotic cell death of the CD95-carrying cells. Further, a monoclonal antibody has been prepared that is specific for CD95, this monoclonal antibody being capable of inducing apoptotic cell death in cells carrying CD95 including mouse cells transformed by cDNA encoding human CD95 (Itoh et al, 1991).
While some of the cytotoxic effects of lymphocytes are mediated by interaction of a lymphocyte-produced ligand with the widely occurring cell surface receptor CD95, which has the ability to trigger cell death, it has also been found that various other normal cells, besides T lymphocytes, express CD95 on their surface and can be killed by the triggering of this receptor. Uncontrolled induction of such a killing process is suspected to contribute to tissue damage in certain diseases, for example, the destruction of liver cells in acute hepatitis. Accordingly finding ways to restrain the cytotoxic activity of CD95 may have therapeutic potential.
Conversely, since it has also been found that certain malignant cells and HIV-infected cells carry CD95 on their surface, antibodies against CD95, or the CD95 ligand, may be used to trigger the CD95 mediated cytotoxic effects in these cells and thereby provide a means for combating such malignant cells or HIV-infected cells (see Itoh et al, 1991). Finding yet other ways for enhancing the cytotoxic activity of CD95 may therefore also have therapeutic potential.
It has been a long felt need to provide a way for modulating the cellular response to TNF (alpha or beta) and CD95 ligand. For example, in the pathological situations mentioned above, where TNF or CD95 ligand is overexpressed, it is desirable to inhibit the TNF- or CD95 ligand-induced cytocidal effects, while in other situations, e.g., wound healing applications, it is desirable to enhance the TNF effect, or in the case of CD95, in tumor cells or HIV-infected cells, it is desirable to enhance the CD95 mediated effect.
A number of approaches have been made by the applicants (see, for example, European Application Nos. EP 186833, EP 308378, EP 398327 and EP 412486) to regulate the deleterious effects of TNF by inhibiting the binding of TNF to its receptors using anti-TNF antibodies or by using soluble TNF receptors (being essentially the soluble extracellular domains of the receptors) to compete with the binding of TNF to the cell surface-bound TNF-Rs. Further, on the basis that TNF-binding to its receptors is required for the TNF-induced cellular effects, approaches by applicants (sec for example EP 568,925) have been made to modulate the TNF effect by modulating the activity of the TNF-Rs.
For example, EP 568925 relates to a method of modulating signal transduction and/or cleavage in TNF-Rs whereby peptides or other molecules may interact either with the receptor itself or with effector proteins interacting with the receptor, thus modulating the normal function of the TNF-Rs. In EP 568925, there is described the construction and characterization of various mutant forms of CD120a, having mutations in its extracellular, transmembrane, and intracellular domains. In this way, regions within the above domains of CD120a were identified as being essential to the functioning of the receptor, i.e., the binding of the ligand (TNF) and the subsequent signal transduction and intracellular signaling which ultimately results in the observed TNF-effect on the cells. Further, there is also described a number of approaches to isolate and identify proteins, peptides or other factors which are capable of binding to the various regions in the above domains of CD120a, which proteins, peptides and other factors may be involved in regulating or modulating the activity of TNF-Rs. A number of approaches for isolating and cloning the DNA sequences encoding such proteins and peptides; for constructing expression vectors for the production of these proteins and peptides; and for the preparation of antibodies or fragments thereof which interact with CD120a or with the above proteins and peptides that bind various regions of CD120a are also set forth in EPO 368925. However, EP 568925 does not specify the actual proteins and peptides which bind to the intracellular domains of the TNF-Rs (e.g., CD95), nor does it describe the yeast two-hybrid approach to isolate and identify such proteins or peptides which bind to the intracellular domains of TNF-Rs. Similarly, in EP 568925 there is no disclosure of proteins or peptides capable of binding the intracellular domain of CD95.
Thus, when it is desired to inhibit the effect of TNF, or of the CD95 ligand, it would be desirable to decrease the amount or the activity of TNF-Rs or CD95 at the cell surface, while an increase in the amount or the activity of TNF-Rs or CD95 would be desired when an enhanced TNF or CD95 ligand effect is sought. To this end the promoters of both the CD120a and the CD120b have been sequenced, analyzed and a number of key sequence motifs have been found that are specific to various transcription regulating factors, and as such the expression of these TNF-Rs can be controlled at their promoter level, i.e., inhibition of transcription from the promoters for a decrease in the number of receptors, and an enhancement of transcription from the promoters for an increase in the number of receptors (EP 606869 and WO 9531206).
While it is known that the tumor necrosis factor (TNF) receptors, and the structurally-related receptor CD95, trigger in cells, upon stimulation by leukocyte-produced ligands, destructive activities that lead to their own demise, the mechanisms of this triggering are still little understood. Mutational studies indicate that in CD95 and CD120a signaling for cytotoxicity involve distinct regions within their intracellular domains (Brakebusch et al, 1992; Tartaglia et al, 1993, Itoh and Nagata, 1993). These regions (the ‘death domains’) have sequence similarity. The ‘death domains’ of both CD95 and CD120a tend to self-associate. Their self-association apparently promotes the receptor aggregation which is necessary for initiation of signaling (see Song et al, 1994; Wallach et al, 1994; Boldin et al, 1995), and at high levels of receptor expression can result in triggering of ligand-independent signaling (Boldin et al, 1995).
Some of the cytotoxic effects of lymphocytes are mediated by interaction of a lymphocyte-produced ligand with CD95, a widely occurring cell surface receptor which has the ability to trigger cell death (see also Nagata and Goldstein, 1995); and that cell killing by mononuclear phagocytes involves a ligand-receptor couple, TNF and its receptor CD120a that is structurally related to CD95 and its ligand (see also Vandenabeele et al, 1995). Like other receptor-induced effects, cell death induction by the TNF receptors and CD95 occurs via a series of protein-protein interactions, leading from ligand-receptor binding to the eventual activation of enzymatic effector functions, which in the case studies have elucidated non-enzymatic protein-protein interactions that initiate signaling for cell death: binding of trimeric TNF or the CD95 ligand molecules to the receptors, the resulting interactions of their intracellular domains (Brakebusch et al, 1992; Tartaglia et al, 1993; Itoh and Nagata, 1993) augmented by a propensity of the death-domain motifs to self-associate (Boldin et al, 1995a), and induced binding of two cytoplasmic proteins (which can also bind to each other),to the receptors' intracellular domains—MORT-1 (or FADD) to CD95 (Boldin et al, 1995b; Chinnaiyan et al, 1995; Kischkel et al, 1995) and TRADD to CD120a (Hsu et al, 1995; Hsu et al, 1996). Three proteins that bind to the intracellular domain of CD95 and CD120a at the ‘death domain’ region involved in cell-death induction by the receptors through hetero-association of homologous regions and that independently are also capable of triggering cell death were identified by the yeast two-hybrid screening procedure. One of these is the protein, MORT-1 (Boldin et al 1995b), also known as FADD (Chinnaiyan et al, 1995) that binds specifically to CD95. The second one, TRADD (see also Hsu et al, 1995, 1996), binds to CD120a, and the third, RIP (see also Stanger et al, 1995), binds to both CD95 and CD120a. Besides their binding to CD95 and CD 120a, these proteins are also capable of binding to each other, which provides for a functional “cross-talk” between CD95 and CD120a. These bindings occur through a conserved sequence motif, the ‘death domain module’ common to the receptors and their associated proteins. Furthermore, although in the yeast two-hybrid test MORT-1 was shown to bind spontaneously to CD95, in mammalian cells, this binding takes place only after stimulation of the receptor, suggesting that MORT-1 participates in the initiating events of CD95 signaling. MORT-1 does not contain any sequence motif characteristic of enzymatic activity, and therefore, its ability to trigger cell death seems not to involve an intrinsic activity of MORT-1 itself, but rather, activation of some other protein(s) that bind MORT-1 and act further downstream in the signaling cascade. Cellular expression of MORT-1 mutants lacking the N-terminal part of the molecule have been shown to block cytotoxicity induction by CD95 or CD120a (Hsu et al, 1996; Chinnaiyan et al, 1996), indicating that this N-terminal region transmits the signaling for the cytocidal effect of both receptors through protein-protein interactions.
Recent studies have implicated a group of cytoplasmic thiol proteases which are structurally related to the Caenorhabditis elegans protease CED3 and to the mammalian interleukin-1 beta converting enzyme (ICE) in the onset of various physiological cell death processes (reviewed in Kumar, 1995 and Henkart, 1996). There have also been some indications that protease(s) of this family may take part in the cell-cytotoxicity induced by CD95 and TNF-Rs. Specific peptide inhibitors of the proteases and two virus-encoded proteins that block their function, the cowpox protein crmA and the Baculovirus p35 protein, were found to provide protection to cells against this cell-cytotoxicity (Enari et al, 1995; Los et al, 1995; Tewari et al, 1995; Xue et al, 1995; Beidler et al, 1995). Rapid cleavage of certain specific cellular proteins, apparently mediated by protease(s) of the CED3/ICE family, could be demonstrated in cells shortly after stimulation of CD95 or TNF-Rs.
One such protease and various isoforms thereof (including inhibitory ones), is known as MACH (now caspase-8) which is a MORT-1 binding protein and which serves to modulate the activity of MORT-1 and hence, of CD95 and CD120a, and which may also act independently of MORT-1, has been recently isolated, cloned, characterized, and its possible uses also described, as is set forth in detail and incorporated herein in their entirety by reference, in co-owned, copending Israel Patent Application Nos. IL 114615, 114986, 115319, 116588 and 117932, as well as their corresponding PCT counterpart No. PCT/US96/10521, and in a recent publication of the present inventors (Boldin et al, 1996). Another such protease and various isoforms thereof (including inhibitory ones), designated Mch4 (also called caspase-10) has also recently been isolated and characterized by the present inventors (unpublished) and others (Fernandes-Alnemri et al, 1996; Srinivasula et al, 1996). Caspase-10 is also a MORT-1 binding protein which serves to modulate the activity of MORT-1 and hence likely also of CD95 and CD120a, and which may also act independently of MORT-1. Thus, details concerning all aspects, features, characteristics and uses of caspase-10 are set forth in the above noted publications, all of which are incorporated herein in their entirety by reference.
It should also be noted that the caspases, caspase-8 and caspase-10, which have similar prodomains (see Boldin et al, 1996; Muzio et al, 1996; Fernandes-Alnemri et al, 1996; Vincent and Dixit, 1997) interact through their prodomains with MORT-1, this interaction being via the ‘death domain motif’ or ‘death effector domain’, DED, present in the N-terminal part of MORT-1 and present in duplicate in caspase-8 and caspase-10 (see Boldin et al, 1995b; Chinnalyan et al, 1995).
Such proteases, now known as caspases (cysteine aspartate-specific proteinases), are a growing family of cysteine proteases that share several common features. Most of the caspases have been found to participate in the initiation and execution of programmed cell death or apoptosis, while the others appear to be involved in the production of proinflammatory cytokines (Nicholson, D W et al 1997; Salvesen, G S et al 1997; Cohen, G M 1997) They are synthesized as catalytically inactive precursors and are generally activated by cleavage after specific internal aspartate residues present in interdomain linkers. The cleavage sites of caspases are defined by tetrapeptide sequences (X-X-X-D) and cleavage always occurs downstream of the aspartic acid. As a result certain mature active caspases can process and activate their own as well as other inactive precursors (Fernandes-Alnemri, T et al 1996, Srinivasula, S M et al 1996).
Activation of the programmed cell death process is generally specific and involves sequential processing of downstream caspases named “execution” caspases by upstream caspases named “initiator” caspases. The functional characteristics of the two classes of caspases are also reflected by their structure. In fact the “initiator caspases” contain longer prodomain regions as compared to the “executioner” caspases (Salvesen, GS et al 1997, Cohen, G M 1997). The long prodomain allows the initiator or “'apical” caspases to be activated by triggering of the death receptors of the TNF receptor family. Upon ligand-induced trimerization of the death receptors, the initiator caspases are recruited through their long N-terminal prodomain to interact with specific adapter molecules to form the death inducing signaling complex (Cohen, G M 1997, Kischkel, F C et al 1995). For example, caspase-8/MACH and probably caspase-10, which contain two Death Effector Domains (DED) or FADD domains, are recruited to the receptor complex by the adapter molecules FADD/MORT-1, whereas caspase-2 is recruited by CRADD/RAIDD and RIP (Nagata, S et al 1997; MacFarlane, M et al 1997; Ahmad, M et al 1997; Duan, H et al 1997). Due to the trimeric nature of the activated receptor complex at least two caspase molecules are thought to be brought in close proximity to each other thus leading to their activation by autocatalytic processing (Yang et al 1998, Muzio et al 1998).
Caspases are synthesized as proenzymes consisting of three major subunits, the N-terminal prodomain, and two subunits, which are sometimes separated by a linker peptide. The two subunits have been termed “long” or subunit 1 containing the active enzymatic site, and “short” or subunit 2. For full activation of the enzyme, the prodomain and the two subdomains are cleaved. The two cleaved subunits form a heterodimer, whereby the long domain is derived from the N-terminus, and the short subunit is derived from the C-terminal region of the caspase precursor. Based on the deduced three dimensional structure of caspase-3, it appears that the C-terminal end of the long domain as well as the N-terminus of the short subdomain have to be freed and the C-terminus of the short subunit has to be brought into close proximity with the N-terminus of the long subunit in order to yield a correctly folded and active enzyme (Rotonda et al 1996, Mittl et al 1997, Srinivasula et al 1998).
N-acetylglucosamine-6-phosphate deacetylase is an intracellular enzyme known to be involved in the intracellular metabolism of glucosamine. A genomic DNA fragment containing N-acetylglucosamine-6-phosphate deacetylase was cloned from the chitinase-producing bacterium Vibrio cholerae (Yamano et al, Biosci Biotechnol Biochem. 61:1349-53 (1997)).
The nagA gene encoding E. coli N-acetylglucosamine-6-phosphate deacetylase is also known [see, e.g., Peri et al, 68(1):123-137 (1990)]. Human N-acetylglucosamine-6-phosphate deacetylase has so far not yet been reported as cloned and sequenced.